Enzymes with improved phytase activity

ABSTRACT

The present invention provides phosphatases with improved phytase activity. The invention provides proteolytic fragments of phosphatase having improved phytase activity. A recombinant gene encoding a phosphatase fragment having improved phytase activity is also provided. The invention also includes a method of increasing the phytase activity of phosphatase by treating the phosphatase with a protease. In addition, the invention provides a new phosphatase, AppA2, having improved properties.

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/127,032, filed Mar. 31, 1999.

BACKGROUND OF THE INVENTION

Phytases are myo-inositol hexakisphosphate phosphohydrolases thatcatalyze the stepwise removal of inorganic orthophosphate from phytate(myo-inositol hexakisphosphate) (1). There are two types of phytases.One is called 3-phytase (EC.3.1.3.8) which initiates the removal ofphosphate groups at the positions 1 and 3 of the myo-inositol ring. Theother is called 6-phytase (EC.3.1.3.26) which first frees the phosphateat the position 6 of the ring. While no phytase has been identified fromanimal tissues, plants usually contain 6-phytases and a broad range ofmicroorganisms, including bacteria, filamentous fungi, and yeast,produce 3-phytases (2-9). Because over 70% of the total phosphorus infoods or feeds of plant origin is in the form of phytate that is poorlyavailable to simple-stomached animals and humans, phytases are of greatuses in improving mineral nutrition of these species (10-16).Supplemental microbial phytases in diets for swine and poultryeffectively enhance bioavailability of phytate phosphorus and reduce theneed for inorganic phosphorus supplementation (11-15), resulting lessphosphorus pollution in areas of intensive animal production (8-15).However, a relatively high level of phytase supplementation is necessaryin animal diets (10-16), because a considerable amount of the enzyme isdegraded in stomach and small intestine (13), probably by proteolysis ofpepsin and trypsin. Meanwhile, the proteolytic profiles of variousphytases were not studied. Clearly, a better understanding of theirsensitivities to trypsin and pepsin hydrolysis could be helpful forimproving the nutritional value of phytases. Aspergillus niger phytasegene (phyA) has been overexpressed in its original host (17) and therecombinant enzyme (r-PhyA, EC 3.1.3.8) has been used in animal diets asa commercial phytase (13, 14). This enzyme is a glycoprotein ofapproximately 80 kDa. Escherichia coli pH 2.5 acid phosphatase gene(appA) has also been characterized (18, 19). Animal experiments havedemonstrated that the recombinant enzyme (r-AppA, EC: 3.1.3.2) is aseffective as r-PhyA in releasing phytate phosphorus in animal diets(14).

But, expenses of the limited available commercial phytase supply and theactivity instability of the enzyme to heat of feed pelleting precludeits practical use in animal industry. Therefore, there is a need forenzymes which have a high level of phytase activity and a high level ofstability for use in animal feed.

SUMMARY OF THE INVENTION

The present invention provides a phosphatase fragment having improvedphytase activity. A fragment of a phosphatase having increased phytaseactivity is produced by treating the phosphatase with a protease.

The invention further provides a recombinant gene encoding a phosphatasefragment having improved phytase activity. The vector consists of apromoter, a coding region encoding the phosphatase fragment, and aterminator.

In another embodiment, the invention provides a method of increasing thephytase activity of phosphatase by treating the phosphatase with aprotease.

The invention also provides a phosphatase having improved phytaseactivity, which has an amino acid sequence as shown in SEQ. ID No. 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the change in phytase activity after protease digestion.FIG. 1A shows phytase activity changes of r-PhyA and r-AppA incubatedwith different ratios of trypsin/protein (w/w) (r=0.001, 0.005, 0.01,and 0.025). Symbols: r-PhyA (▪) and r-AppA (). The results are themean±SEM from five independent experiments. * indicates statisticalsignificance (P<0.01) versus untreated r-PhyA or r-AppA control. FIG. 1Bshows phytase activity changes of r-PhyA and r-AppA incubated withdifferent ratios of pepsin/protein (w/w) (r=0.001, 0.002, 0.005, and0.01. Symbols: r-PhyA (□) and r-AppA (∘). The results are the mean±SEMfrom seven independent experiments. * indicates statistical significance(P<0.01) versus untreated r-PhyA or r-AppA control.

FIG. 2 shows residual phytase activity of r-PhyA and r-AppA aftertrypsin or pepsin hydrolysis during a time course (0, 1, 51 30, and 120min). Symbols: trypsin-digested r-PhyA (▪) or r-AppA ();pepsin-digested r-PhyA (□) and r-AppA (∘). The ratio of trypsin/phytase(w/w) used was: r=0.01 (w/w). The ratio of pepsin/phytase used was:r=0.005. The results are the mean±SEM from six independentexperiments. * indicates statistical significance (P<0.01) versusuntreated r-PhyA or r-AppA control.

FIG. 3 shows the results of SDS-polyacrylamide gel electrophoresis ofr-AppA (12%, Panel A) or r-PhyA (20%, Panel B) digested products bydifferent amounts of trypsin (r=0.001, 0.00 5, 0.01, and 0.025, (w/w).Proteins were stained using Coomasie blue. T: trypsin control, C:purified r-AppA (Panel A) or r-PhyA (Panel B). The protein marker (M) isa 10 kDa ladder [10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, and200 kDa) (Gibco). The results are representative from four independentexperiments.

FIG. 4 shows the results from SDS-polyacrylamide gel (20%)electrophoresis of r-AppA (FIG. 4A) or r-PhyA (FIG. 4B) digestedproducts by different amounts of pepsin (r=0.001, 0.002, 0.005, and0.01, (w/w)). Proteins were stained using Coomasie blue. T: trypsincontrol, C: purified r-AppA (FIG. 4A) or r-PhyA (FIG. 4B). The proteinmarker (M) is a 10 kDa ladder [10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, and 200 kDa) (Gibco). The results are representative from sixindependent experiments.

FIG. 5 shows the amounts of inorganic phosphorus (iP) released fromsoybean meal by r-PhyA and r-AppA incubated with differentconcentrations of trypsin (r=0.001, 0.005, 0.01, and 0.025, w/w) (FIG.5A), or pepsin (r=0.001, 0.002, 0.005, and 0.01) (FIG. 5B). Symbols:r-AppA (▪), r-PhyA (□). The results are the mean SEM from threeindependent experiments. * indicates statistical significance (P<0.01)versus untreated r-AppA or r-PhyA control.

FIG. 6 shows the nucleotide sequence of the appA2 gene and its deducedamino acid sequence. The untranslated region is indicated by lowercaseletters. The underlined sequences are the primers used to amplifyappA2′(Pf1: 1-22, and K2: 1467-1480), appA2 (E2: 247-26 1, and K2:1467-1480). Potential N-glycosylation sites are boxed. The sequence ofappA2 has been transmitted to Genebank data library with accessionnumber 250016. The appA2′ gene is formed by nucleotides 1-1489 (SEQ. ID.No. 9). The deduced amino acid sequence of the appA2 gene is identifiedas amino acids 1-433 (SEQ. ID. No. 1), corresponding to nucleic acidbases 182-1480 of the appA2′ gene. A lead amino acid sequence encoded bythe appA2 gene is identified as amino acids 1-30 (i.e., SEQ. ID. No. 8)of the first numbered amino acid sequence, and corresponds to nucleicacid bases 16-105 of SEQ. ID. No. 9.

FIG. 7 is a time course of extracellular phytase (□) and acidphosphatase (∘) activities, and CIPPA2 mRNA expression (▴) in Pichiapastoris transformed with appA2 after induction. Results are expressedas the mean±SEM from three experiments.

FIG. 8 shows a northern blot analysis of appA2 mRNA expression in Pichiapastoris transformed with appA2 after induction (FIG. 8A). Hybridizationwas realized using [α-³²P] labeled appA2 as a probe. Twenty μg of totalRNA was loaded per lane. Panel B represents the equal RNA loadingvisualized by the yeast rRNA under UV.

FIG. 9 shows the pH dependence of the enzymatic activity at 37° C. ofthe purified r-appA2 (), r-appA (▴), and r-phyA (□) with sodium phytateas the substrate. Buffers: pH 1.5-4.5, 0.2M glycine-HCl; pH 5.5-7.5, 0.2M citrate; pH 8.5-11, 0.2 M Tris-HCl. Results are expressed as the meanSEM from three experiments.

FIG. 10 shows a non-denaturing gel (15%) electrophoresis analysis of theremaining acid phosphatase activity of r-appA2 after incubated atdifferent temperatures for 20 min. After the heat treatment, the sampleswere put on ice for 5 min before being loaded onto the gel (200 μgprotein/lane).

FIG. 11 shows the hydrolysis of phytate phosphorus in soybean meal bydifferent amounts (100, 300, 600, and 900 PU) of purified r-appA2 (),r-appA (▴), and r-phyA (□) enzymes. * indicates significant differences(P<0.05) between r-appA2 and other two enzymes. Results are expressed asthe mean±SEM from three experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides phosphatases having improved phytaseactivity.

One embodiment of the invention provides a phosphatase fragment havingimproved phytase activity. The phosphatase is treated with a proteaseand fragments having phosphatase activity are selected. As discussed infurther detail below, these fragments, have improved phytase activitycompared to the full length peptide.

In a preferred embodiment, the protease is pepsin.

In addition to producing the active fragment by proteolysis of the fulllength peptide, the present invention also provides a recombinant genehaving a promoter, a coding region encoding the phosphatase fragmentaccording to claim 1, and a terminator. The recombinant gene can be usedto express the truncated product directly.

The improved phosphatases can by used in animal feed to improve theaccessibility of phosphate to the animal.

In addition to the phosphatase, the invention provides a method ofincreasing the phytase activity of phosphatase by treating thephosphatase with a protease.

In another embodiment, the invention provides a phosphatase havingimproved phytase activity, which has an amino acid sequence as shown inSEQ. ID No. 1 as shown in FIG. 6.

Preferably, the protein or polypeptide with phytase activity is secretedby the cell into growth media. This allows for higher expression levelsand easier isolation of the product. The protein or polypeptide withphytase activity is coupled to a signal sequence capable of directingthe protein out of the cell. Preferably, the signal sequence is cleavedfrom the protein.

In a preferred embodiment, the heterologous gene, which encodes aprotein or polypeptide with phytase activity, is spliced in frame with atranscriptional enhancer element.

A preferred phosphatase is encoded by the appA gene of E. coli. Thegene, originally defined as E. coli periplasmic phosphoanhydridephosphohydrolase (appA) gene, contains 1,298 nucleotides (GeneBankaccession number: M58708). The gene was first found to code for an acidphosphatase protein of optimal pH of 2.5 (EcAP) in E. coli. The acidphosphatase is a monomer with a molecular mass of 44,644 daltons. MatureEcAP contains 410 amino acids (18). Ostanin, et al. overexpressed appAin E. coli BL21 using a pT7 vector and increased its acid phosphataseactivity by approximately 400-folds (440 mU/mg protein) (20). Theproduct of the appA gene was not previously known to have phytaseactivity.

The phosphatase can be expressed in any prokaryotic or eukaryoticexpression system. A variety of host-vector systems may be utilized toexpress the protein-encoding sequence(s). Preferred vectors include aviral vector, plasmid, cosmid or an oligonucleotide. Primarily, thevector system must be compatible with the host cell used. Host-vectorsystems include but are not limited to the following: bacteriatransformed with bacteriophage DNA, plasmid DNA, or cosmid DNA;microorganisms such as yeast containing yeast vectors; mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus); and plantcells infected by bacteria. The expression elements of these vectorsvary in their strength and specificities. Depending upon the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements can be used.

Preferred hosts for expressing phosphatase include fungal cells,including species of yeast or filamentous fungi, may be used as hostcells in accordance with the present invention. Preferred yeast hostcells include different strains of Saccharomyces cerevisiae. Otheryeasts like Kluyveromyces, Torulaspora, and Schizosaccharomyces can alsobe used. In a preferred embodiment, the yeast strain used to overexpressthe protein is Saccharomyces cerevisiae. Filamentous fungi host cellsinclude Aspergillus and Neurospora.

In another embodiment of the present invention, the yeast strain is amethylotrophic yeast strain. Methylotrophic yeast are those yeast generacapable of utilizing methanol as a carbon source for the production ofthe energy resources necessary to maintain cellular function andcontaining a gene for the expression of alcohol oxidase. Typicalmethylotrophic yeasts include members of the genera Pichia, Hansenula,Torulopsis, Candida, and Karwinskia. These yeast genera can use methanolas a sole carbon source. In a preferred embodiment, the methylotrophicyeast strain is Pichia pastoris.

A preferred embodiment of the invention is a protein or polypeptidehaving phytase activity with optimum activity in a temperature range of57 to 65° C. A more preferred embodiment is a protein or polypeptidehaving phytase activity, where its temperature range for optimumactivity is from 58 to 62° C.

Yet another preferred embodiment is a protein or polypeptide havingphytase activity where the protein retains at least 40% of its activityafter heating the protein for 15 minutes at 80° C. More preferred is aprotein or polypeptide having phytase activity where the protein retainsat least 60% of its activity after heating the protein for 15 minutes at60° C.

Purified protein may be obtained by several methods. The protein orpolypeptide of the present invention is preferably produced in purifiedform (preferably at least about 80%, more preferably 90%, pure) byconventional techniques. Typically, the protein or polypeptide of thepresent invention is secreted into the growth medium of recombinant hostcells. Alternatively, the protein or polypeptide of the presentinvention is produced but not secreted into growth medium. In suchcases, to isolate the protein, the host cell carrying a recombinantplasmid is propagated, lysed by sonication, heat, or chemical treatment,and the homogenate is centrifuged to remove cell debris. The supernatantis then subjected to sequential ammonium sulfate precipitation. Thefraction containing the polypeptide or protein of the present inventionis subjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the proteins. If necessary, theprotein fraction may be further purified by HPLC.

The present invention also provides a yeast strain having a heterologousgene which encodes a protein or polypeptide with phytase activity. Theheterologous gene should be functionally linked to a promoter capable ofexpressing phytase in yeast and followed by a transcriptionalterminator.

Yet another aspect of the invention is a vector for expressing phytasein a host. The vector carries a phosphatase gene which encodes a proteinor polypeptide with phytase activity.

For cloning into yeast, the gene can be cloned into any vector whichreplicates autonomously or integrates into the genome of yeast. The copynumber of autonomously replicating plasmids, e.g. YEp plasmids may behigh, but their mitotic stability may be insufficient (48). They maycontain the 2 mu-plasmid sequence responsible for autonomousreplication, and an E. coli sequence responsible for replication in E.coli. The vectors preferably contain a genetic marker for selection ofyeast transformants, and an antibiotic resistance gene for selection inE. coli. The episomal vectors containing the ARS and CEN sequences occuras a single copy per cell, and they are more stable than the YEpvectors. Integrative vectors are used when a DNA fragment is integratedas one or multiple copies into the yeast genome. In this case, therecombinant DNA is stable and no selection is needed (49-51). Somevectors have an origin of replication, which functions in the selectedhost cell. Suitable origins of replication include 2μ, ARS1, and 25 μM.The vectors have restriction endonuclease sites for insertion of thefusion gene and promoter sequences, and selection markers. The vectorsmay be modified by removal or addition of restriction sites, or removalof other unwanted nucleotides.

The phytase gene can be placed under the control of any promoter (52).One can choose a constitutive or regulated yeast promoter. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (53) or other glycolyticenzymes (54), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in EP A-73,657 to Hitzeman, which ishereby incorporated by reference. Another alternative is theglucose-repressible ADH2 promoter (56, 57), which are herebyincorporated by reference.

One can choose a constitutive or regulated yeast promoter. The strongpromoters of e.g., phosphoglycerate kinase (PGK) gene, other genesencoding glycolytic enzymes, and the alpha -factor gene, areconstitutive. When a constitutive promoter is used, the product issynthesized during cell growth. The ADH2 promoter is regulated withethanol and glucose, the GAL-1-10 and GAL7 promoters with galactose andglucose, the PHO5 promoter with phosphate, and the metallothioninepromoter with copper. The heat shock promoters, to which the HSP150promoter belongs, are regulated by temperature. Hybrid promoters canalso be used. A regulated promoter is used when continuous expression ofthe desired product is harmful for the host cells. Instead of yeastpromoters, a strong prokaryotic promoter such as the T7 promoter, can beused, but in this case the yeast strain has to be transformed with agene encoding the respective polymerase. For transcription termination,the HSP150 terminator, or any other functional terminator is used. Here,promoters and terminators are called control elements. The presentinvention is not restricted to any specific vector, promoter, orterminator.

The vector may also carry a selectable marker. Selectable markers areoften antibiotic resistance genes or genes capable of complementingstrains of yeast having well characterized metabolic deficiencies, suchas tryptophan or histidine deficient mutants. Preferred selectablemarkers include URA3, LEU2, HIS3, TRP1, HIS4, ARG4, or antibioticresistance genes.

The vector may also have an origin of replication capable of replicationin a bacterial cell. Manipulation of vectors is more efficient inbacterial strains. Preferred bacterial origin of replications are ColE1,Ori, or oriT.

A leader sequence either from the yeast or from phytase genes or othersources can be used to support the secretion of expressed phytase enzymeinto the medium. The present invention is not restricted to any specifictype of leader sequence or signal peptide.

Suitable leader sequences include the yeast alpha factor leadersequence, which may be employed to direct secretion of the phytase. Thealpha factor leader sequence is often inserted between the promotersequence and the structural gene sequence (58; U.S. Pat. No. 4,546,082;and European published patent application No. 324,274, which are herebyincorporated by reference). Another suitable leader sequence is the S.cerevisiae MF alpha 1 (alpha-factor) is synthesized as a prepro form of165 amino acids comprising signal-or prepeptide of 19 amino acidsfollowed by a “leader” or propeptide of 64 amino acids, encompassingthree N-linked glycosylation sites followed by (LysArg(Asp/Glu, Ala)2-3alpha-factor)4 (58). The signal-leader part of the preproMF alpha 1 hasbeen widely employed to obtain synthesis and secretion of heterologousproteins in S. cerivisiae. Use of signal/leader peptides homologous toyeast is known from. U.S. Pat. No. 4,546,082, European PatentApplications Nos. 116,201; 123,294; 123,544; 163,529; and 123,289 and DKPatent Application No. 3614/83, which are hereby incorporated byreference. In European Patent Application No. 123,289, which is herebyincorporated by reference, utilization of the S. cerevisiae a-factorprecursor is described whereas WO 84/01153, which is hereby incorporatedby reference, indicates utilization of the Saccharomyces cerevisiaeinvertase signal peptide, and German Patent Application DK 3614/83,which is hereby incorporated by reference, indicates utilization of theSacchalomyces cerevisiae PH05 signal peptide for secretion of foreignproteins.

The alpha -factor signal-leader from Saccharomyces cerevisiae (MF alpha1 or MF alpha 2) may also be utilized in the secretion process ofexpressed heterologous proteins in yeast (U.S. Pat. No. 4,546,082,European Patent Applications Nos. 16,201; 123,294; 123 544; and 163,529,which are hereby incorporated by reference). By fusing a DNA sequenceencoding the S. cerevisiea MF alpha 1 signal/leader sequence at the 5′end of the gene for the desired protein secretion and processing of thedesired protein was demonstrated. The use of the mouse salivary amylasesignal peptide (or a mutant thereof) to provide secretion ofheterologous proteins expressed in yeast has been described in PublishedPCT Applications Nos. WO 89/02463 and WO 90/10075, which are herebyincorporated by reference.

U.S. Pat. No. 5,726,038 describes the use of the signal peptide of theyeast aspartic protease 3, which is capable of providing improvedsecretion of proteins expressed in yeast. Other leader sequencessuitable for facilitating secretion of recombinant polypeptides fromyeast hosts are known to those of skill in the art. A leader sequencemay be modified near its 3′ end to contain one or more restrictionsites. This will facilitate fusion of the leader sequence to thestructural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described in Hinnen et al. (59). The Hinnen et al.protocol selects for Trp transformants in a selective medium, whereinthe selective medium consists of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil.

The gene may be maintained on stable expression vector, an artificialchromosome, or by integration into the yeast host cell chromosome.Integration into the chromosome may be accomplished by cloning thephytase gene into a vector which will recombine into a yeast chromosome.Suitable vectors may include nucleotide sequences which are homologousto nucleotide sequences in the yeast chromosome. Alternatively, thephytase gene may be located between recombination sites, such astransposable elements, which can mobilize the gene into the chromosome.

The present invention also provides a method of producing phytase byproviding an isolated phosphatase gene, which encodes a protein orpolypeptide with phytase activity, and expressing the gene in host cell.The phosphatase preferably is a microbial phosphatase. In a morepreferred embodiment, the microbial phosphatase is an Escherichia coliphosphatase. Also preferred are the microbial phosphatases, AppA andAppA2.

A method of converting phytate to inositol and inorganic phosphorus isalso provided. An appA gene is isolated from an organism, usingtechniques well known in the art. A protein or polypeptide with phytaseactivity is then expressed from the gene in a host cell. The resultingprotein or polypeptide is mixed or contacted with phyate. This techniqueis especially useful for treating phytate in food or animal feed.

The preferred appA gene is isolated from Escherichia coli.

While the phytase enzyme produced in a yeast system released phytate-Pfrom corn and soy as effectively as the currently commercial phytase, itappeared to be more thermostable. This phytase overexpression system inyeast can be used to provide thermostable phytase for use in the foodand feed industries.

EXAMPLES Example 1 Materials and Methods for Examples 2-6

Expression of r-AppA. The appA gene (Genebank accession number M58708)was obtained from E. coli BL21 strain transformed by an expressionvector pAPPA1 (20). A 1.35 kb DNA fragment containing the coding regionof appA was amplified by PCR following the manufacturer instructions(Perkin Elmer). Primers were derived from 5′ and 3′ regions of thenucleotide sequence (18), and include: E2 [forward: 242-252]:

5′GGAATTCCAGAGTGAGCCGGA3′ (SEQ. ID. No. 2) and K2 [reverse: 1468-1490]:5′GGGGTACCTTACAAACTGCACG3′ (SEQ. ID. No. 3). These two primers weresynthesized by the Cornell University Oligonucleotide Synthesis Facility(Ithaca, N.Y.). The amplified product was sliced from a 1% agarose gel,and eluted with GENECLEAN II kit (Bio101). The purified fragment wasfirst cloned into pGEM T-easy vector (Promega), and then inserted intothe yeast expression vector pPIcZαA (Invitrogen) at EcoRI site. E. colistrain TOP10F′ (Invitrogen) was used as an initial host to amplify thesetwo constructs. The pPIcZtαA vector containing appA was transformed intoP. pastoris strain X33 by electroporation according to themanufacturer's instructions (Invitrogen). The transformed cells wereplated into YPD-Zeocin agar medium and positive colonies were incubatedin minimal media with glycerol (BMGY) for 24 h. When the yeast celldensity reached 2.5×10⁸ cells/ml (OD₆₀₀=5), the cells were centrifugedand suspended in 0.5% methanol medium (BMMY) to induce the appA geneexpression. Total yeast genomic DNA was extracted from the transformedX33 cells after induction and used as a template to check the presenceof the appA gene by PCR using the same primers as described above. Theamplified DNA fragment was sequenced at the Cornell University DNAServices-Facility using Taq Cycle automated sequencing with Dye Deoxyterminators (Applied Biosystems, Forster City, Calif.).

Purification of r-PhyA and r-AppA. r-PhyA was obtained from BASF (MtOlive, N.J.). Both r-PhyA and r-AppA enzymes were initially suspendedinto 50 mM Tris-HCl, pH 7, and ammonium sulfate was added to 25% ofsaturation. After the mixture was centrifuged (25,000 g, 20 min), thesupernatant was saved and ammonium sulfate was added to 75% ofsaturation. Then, the mixture was centrifuged (25,000 g, 20 min) and thepellet was suspended into 10 mL of 25 mM Tris-HCl, pH 7. The suspensionwas dialyzed overnight against the same buffer and loaded onto aDEAE-Sepharose column (Sigma) equilibrated with 25 mM Tris-HCl, pH 7.Proteins were diluted with 0.2 M NaCl, 25 mM Tris-HCl, pH 7 after thecolumn was washed with 200 n-LL of 25 mM Tris-HCl, pH 7. All thecollected fractions were assayed for phytase activity and proteinconcentration (21). The whole purification was conducted at 4° C., andthe fractions were stored at −20° C. before analysis.

Proteolysis and protein electrophoresis. The purified r-AppA and r-PhyA(2 mg/mL) were incubated with different amounts of pepsin and trypsinfollowing the manufacturer instructions (Sigma). Pepsin (800 units/mgprotein) and trypsin (1,500 BAEE units/mg protein) were dissolved into10 mM HCl, pH 2 (0.1 mg/mL) and 80 mM ammonium bicarbonate, pH 7.5 (0.1mg/mL), respectively. One BAEE unit was defined as 0.001 absorbancechange at 253 nm per minute at pH 7.6 and 250C, with BAEE as asubstrate. In a final volume of 100 μL, 10 μg of purified r-PhyA (0.1PU) or r-AppA (0.08 PU) was incubated with trypsin or pepsin atprotease/phytase (w/w) ratios ranging from 0.001 to 0.01, at 37° C. for1 to 120 min. The reaction was stopped on ice and the pH of the mixturewas adjusted to 8 for protein electrophoresis and phytase activityassay. The digested protein mixtures were analyzed by sodium dodecylsulfate (SDS)-polyacrylamide or urea-SDS-polyacrylamide gelelectrophoresis as previously described (22, 23).

Phytase activity and hydrolysis of phytate phosphorus from soybean meal.Phytase activities of both r-PhyA and r-AppA, prior to or at varioustime points of proteolysis, were determined as previously described(24). The released inorganic phosphorus (1P) was assayed by the methodof Chen et al. (25). One phytase unit (PU) was defined as the activitythat releases 1 μmol of iP from sodium phytate per minute at 37° C. Toconfirm the proteotytic effects of trypsin and pepsin on the residualactivities of r-PhyA and r-AppA, the hydrolysis of phytate phosphorusfrom soybean meal by these two enzymes incubated with different amountsof trypsin or pepsin was monitored. In a 5 mL total reaction, 0.5 mg ofthe purified r-PhyA (5 PU) or r-AppA (4 PU) was incubated with 1 gsoybean meal and pepsin in 10 mM HCl, pH 2.5 or trypsin in 0.2 Mcitrate, pH 6.8 at 3 VC for 2 h. The released iP was determined asdescribed above.

Example 2 Preparation of r-AppA and r-PhyA

Over 30 colonies of X33 transformed with the appA gene expressedextracellular phytase activity that hydrolyzes sodium phytate. Colony 26had the highest activity (88 U/mL) and was chosen for further studies.After the r-PhyA and the r-AppA samples were eluted from theDEAE-Sepharose column, 45 fractions of 4 mL each were collected for bothenzymes to assay for phytase activity. The fractions used forproteolysis had a specific phytase activity of 9.6 and 8.1 U/mg ofprotein for the r-PhyA and r-AppA, respectively.

Example 3 Effects of Trypsin Digestion on the Phytase Activities of BothEnzymes

After 2 hour trypsin digestion, there were significant differences inthe residual phytase activities between the r-PhyA and the r-AppA (FIG.1A). Although both enzymes retained more than 85% of their originalactivities at the trypsin/phytase ratios of 0.001 and 0.005, r-AppA lost64 and 74% of its original activity at the ratio of 0.01 and 0.025,respectively. Meanwhile, r-PhyA lost only 14 and 23% of its originalactivity, respectively. Because of the apparent difference insensitivities of these two enzymes to trypsin digestion at the ratio of0.01, a time course study was conducted with this ratio. Up to 2 hourtrypsin digestion, r-PhyA still retained 90% of its original activity(FIG. 2). In contrast, r-AppA lost 64, 77, 87, and 95% of its originalactivity after 1, 5, 30, and 120 minute digestion, respectively.

Example 4 Effect of Pepsin Digestion on the Phytase Activities of BothEnzymes

After 2 hour pepsin digestion, the residual phytase activity of r-AppAwas totally unexpected. At the ratios of 0.001 and 0.002, the phytaseactivity either remained unchanged or slightly increased. At the ratiosof 0.005 and 0.01, the phytase activity was enhanced by 30% comparedwith the initial value. However, r-PhyA lost 58 and 77% of its originalactivity at these two high ratios (FIG. 1B). Because significantlydifferent responses between r-PhyA and r-AppA at the ratio of 0.005,this ratio was used for a follow-up time course study. There was astepwise increase in phytase activity along with time when the r-AppAwas incubated with pepsin from 0 to 30 min. Thereafter no furtherincrease was observed (FIG. 2). However, r-PhyA lost 42, 73, 82, and 92%of its original activity after 1, 5, 30, and 120 minute incubation,respectively.

Example 5 SDS-polyacrylamide Gel Electrophoresis

When r-AppA was incubated with trypsin, the enzyme protein was degradedat the ratios above 0.01 and was invisible at the ratio of 0.025. Therewas a major band of approximately 28 kDa, with several other bandsbetween this band and the intact protein in the three low ratios oftrypsin. However, that major band was clearly reduced and the otherbands disappeared at the highest ratio of trypsin (FIG. 3A). There weremany intermediary bands when the r-PhyA was incubated with variousamounts of trypsin and there were at least three visible bands at thehighest ratio of trypsin (FIG. 3B). A unique band of approximately 8.4kDa was shown when r-AppA was incubated with pepsin at the ratio above0.002 (FIG. 4A). On the other hand, proteolysis of r-PhyA by variousamounts of pepsin resulted in many diffused and smearing bands, inaddition to a major fragment of approximately 14 kDa (FIG. 4B).

Example 6 Effects of Proteolysis on Phytate-Phosphorus Hydrolysis byr-PhyA and r-AppA

When r-AppA was incubated with soybean meal and different amounts oftrypsin for 2 h at 37° C., the reduction in iP released from soybeanmeal was 3, 13, 34, and 52%, at the ratio of 0.001, 0.005, 0.01, and0.025, respectively (FIG. 5A). Meanwhile, the reduction for r-PhyA atthe same condition was 3, 6, 13, and 28%, respectively. Adding pepsin tor-AppA (ratio 0.005) and the soybean meal mixture resulted inapproximately 30% increase in iP released from soybean meal, comparedwith the control (FIG. 5B). In contrast, the same treatments producedmore than 50% reduction in iP release by r-PhyA.

To date, there have been no specific data on sensitivities of microbialphytases to trypsin and pepsin. In this study, two partially purifiedrecombinant phytases were exposed to single protease digestions, andmeasured the effects of proteolysis on their residual activities andtheir capacity of releasing phytate phosphorus from soybean meal. Theseresults have demonstrated that r-PhyA is more resistant to trypsin andless resistant to pepsin than r-AppA. The proteolytic patterns of thesetwo phytases, shown by SDS-PAGE analysis, are also distinctly different.Presumably, these different susceptibilities to proteases between r-PhyAand r-AppA may be associated with their characteristics of primary aminoacid sequence and peptide folding, because there is a low homology(−15%) of amino acid sequences between these two enzymes (17, 18).However, caution should be given in consideration of the molecularmechanism of phytase proteolysis, which is beyond the original scope ofthe present study. Recent progress in crystallization and (or)preliminary X-ray analysis of the phyA phytase (26) and an E. coliphytase (27) would help us in understanding the structural basis fortheir proteolytic responses.

Unexpectedly, r-AppA showed a 30% increase in residual phytase activityafter pepsin digestion. Likewise, this enzyme also released 30% more iPfrom soybean meal in the presence of pepsin. From the SDS-PAGE analysis,r-AppA was clearly degraded into small peptides by pepsin alongdifferent periods of incubation. Likely, there may be potential pepsinresistant polypeptides with higher phytase activity than the intactr-AppA protein. Although the SDS-PAGE analysis did not offer us anyspecific information on such peptides, pepsin has been shown to convertnatural or synthetic proteins in active polypeptides, such as convertingporcine endothelin to active 21-residue endothelin (28). Pepsin may alsomodulate the structure and functions of certain proteins (29, 30). Asmentioned above, the availability of the recent crystallization data onthe phyA (26) and the E. coli phytases (27) would facilitate targetingsite-directed mutageneses or deletions of the appA gene. Thereby, it maybe possible to unveil the molecular mechanism for the increase ofphytase activity of r-AppA associated with pepsin hydrolysis. In spiteof the biochemical uncertainty of the pepsin resistant r-AppA peptides,this finding has a great nutritional implication. Because pepsin, a welldescribed aspartic protease, is the major protease in the stomach (31),a pepsin resistant phytase polypeptides could allow us to supplement alow level of enzyme to the diets with sufficient activity. Thus, expensefor use of dietary phytase in animal production will be reduced.

It is difficult to compare the activity levels of proteases used in thepresent study with those at the physiological conditions, because the invivo concentrations of pepsin and trypsin have not been well described.An average trypsin activity of 20 to 25 U/mg of protein has beenreported in the intestine of pig (32), which is much higher than thedoses used herein. However, multiple levels of trypsin and pepsin wereused, with 10 to 25 fold range. differences between the lowest andhighest levels. In addition, the iP release from soybean meal by r-AppAor r-PhyA was measured in the presence of pepsin or trypsin, a simulatedin vivo digestive condition. Although both r-AppA and r-PhyA werepartially purified, all the data consistently point toward distinctresponsive patterns of these two recombinant enzymes to pepsin andtrypsin. Thus, this in vitro observation could be relevant tophysiological conditions.

Example 7 Materials and Methods for Examples 8-12

Isolation and identification of phytase producing bacterium colonies.Colon contents were obtained from crossbreed Hampshire-Yorkshire-Durocpigs (13 weeks of age) raised under confinement at Cornell UniversitySwine Farm. These pigs were fed a practical corn-soybean meal diet.Immediately after the pigs were killed, the content of colon was removedby aseptic procedures and kept in anaerobic, sterile plastic bags. A 10g sample was diluted with 190 ml of an anaerobic rumen fluid glucosemedium in a 250 ml rubber-stoppered Erlenmeyer flask. The mixture wasshaken vigorously for 3 min under a CO₂ atmosphere. Serial successivedilutions were made accordingly.

Diluted samples were cultured at 37° C. for 3 days in a modified rumenfluid-glucose-cellobiose-Agar medium containing insoluble calciumphytate (43, 44). Colonies with a clear zone were tested as a potentialproducer of intra and extracellular phytase activity. Phytase activitywas measured using sodium phytate as a substrate (24). One phytase unit(PU) was defined as the activity that releases one μmole of inorganicphosphorus from sodium phytate per minute at 37° C. Acid phosphataseactivity was assayed using p-nitrophenyl phosphate (P-NPP) as asubstrate according to the manufacturer instructions (Sigma, St Louis,Mo.). Identification of the selected colony was conducted in theDiagnostic Laboratory of Cornell Veterinary College (Ithaca, N.Y.).Morphological and physiological characteristics of the isolated colonywere determined by standard procedures.

DNA amplification and sequencing Because the colony that produced thehighest acid phosphatase and phytase activities was identified as an E.coli strain, primers derived from the DNA sequence of E. coli pH 2.5acid phosphatase gene (appA, GeneBank Accession number 145283) (18) wereused to isolate the gene. Primers Pf1 [forward: 1-22]:

5′-TAAGGAGCAGAAACAATGTGGT-3′ (SEQ. ID. No. 4), E2 [forward: 254-264]:

5′-GGAATTCCAGAGTGAGCCGGA-3′ (SEQ. ID. No. 5), and K2 [reverse:1468-1491]:

5′-GGGGTACCTTACAAACTGCACG-3′ (SEQ. ID. No. 6) were synthesized at theCornell University Oligonucleotide Synthesis Facility. The wholesequence and the coding region were amplified using [Pf1-K21 and [E2-K2]primers, respectively. The PCR reaction mixture (100 μl,) contained 500ng of genomic DNA as template, 100 pmole of each primer, 5 U of AmpliTaqDNA polymerase (Perkin Elmer, Norwalk, Conn.), 10 mM Tris-HCl pH 8.3, 50 mM KCl, 12.5 mM MgC 12, and 200 μM each dNTPs (Promega, Madison,Wis.). The reaction was performed by the GeneAmp PCR system 2400 (PerkinElmer). The thermal program included 1 cycle at 94° C. (3 min), 30cycles of [94° C. (0.8 min), 54° C. (1 min) and 72° C. (2 min)] and 1cycle at 72° C. (10 min). Amplified PCR products were resolved by 1% lowmelting agarose (Gibco BRL, Grand Island, N.Y.) gel electrophoresis. Agel slice containing the expected size band was excised and DNA waseluted with GENECLEAN II kit (Bio101, Vista, Calif.). The PCR productswere sequenced at the Cornell University DNA Service Facility using TaqCycle automated sequencing with Dye Deoxy terminators (AppliedBiosystems, Forster City, Calif.). Sequencing experiments were performedfive times and the deduced amino sequence was aligned with other acidphosphatases and phytases using the Multi-align Program CLUSTAL BLAST(45). The two identified PCR fragments [Pf1-K2] and [E2-K2] weredescribed, respectively, as appA2 and appA2 in the following text. Forcomparative purposes, the appA gene was amplified from E. coli BL21(DE3) using the primers [E2-K2]. The PCR reactions and the resultingfragments were processed as described above.

Subcloning and construction of expression vectors. The PCR products[E2-K2] and [Pf1-K2] were cloned into pGEM® T-easy vector (Promega)according to the manufacturer instructions and transformed into TOP10Fto screen for positive colonies. The isolated appA2 and appA fragmentswere inserted into the pPICZαA (Invitrogen, San Diego, Calif.) at theEcoRI and KpnI sites, as described by the manufacturer instruction. Theconstructs were transformed into TOP10F cells which were plated on LBmedium containing 25 μg zeocin/ml. The positive colonies were then grownto prepare DNA for transformation.

Yeast transformation and expression. Pichia pastoris strain X33(Invitrogen) were grown in YPD medium and prepared for transformation,according to the manufacturer instructions. Two μg of plasmid DNA waslinearized using Bg/II and then transformed into Pichia byelectroporation. After incubation for 3 h at 30° C. in 1 M sorbitolwithout agitation, cells were plated in YPD-zeocin agar medium to screenintegration of the transformed gene into the 5′ AOX1 region of the hostchromosomal DNA. After 2 days, transformants were incubated in minimalmedia with glycerol (GMGY medium) for 24 h. After the culture reached adensity of about 2.5 10⁸ cells/ml (OD₆₀₀=5), the cells were spun down(3500 g, 5 min) and then suspended in 0.5% methanol medium (GMMY) toinduce the phytase gene expression.

RNA quantification. Total RNA was extracted from the appA2 transformantsat different times after induction. The RNA was separated in 1%formaldehyde-agarose gel, transferred onto Hybond N+ membrane (AmershamPharmacia Biotech, Piscataway, N.J.) by capillary blotting and UVcross-linked for 2 min. The membrane was then pre-hybridized for 4 h at42° C. The probe was the appA2 [E2-K2] PCR fragment, and was labeledwith [α-³²P]-dCTP (DuPont, Boston, Mass.) using Ready-To-Go TM DNALabeling Beads (Amersham Pharmacia Biotech). The membrane was hybridizedwith the probe overnight at 42° C., and washed three times for 20 min at25° C. and twice at 50° C. in 2×SSC (0.15 M NaCl, 0.015 M sodiumcitrate), 1% sodium dodecyl sulfate (SDS), and finally twice at 50° C.in 0.2×SSC, 0.1% SDS. The autoradiogram was produced by exposing themembrane to an intensifying screen of BAS-III FUJI Imaging plate (Fuji,Japan) for 10 h and quantified using a Bio-Imaging Analyzer (KohshinGraphic Systems, Fuji, Japan). Results were normalized by the relativelevels of 18S rRNA.

Purification of the expressed enzymes. All operations were carried outat 4° C. Both expressed r-appA and r-appA2 enzymes, and the r-phyAphytase expressed in A. niger (kindly provided by BASF, Mt. Olive,N.J.), were suspended in 50 mM Tris-HCl, pH 7 with 25% saturation ofammonium sulfate. The suspension was then centrifuged at 25,000 g for 20min. The supernatant was mixed with 75% saturated ammonium sulfate underagitation for 12 h, and the mixture was centrifuged at 25,000 g for 20min. The pellet was then suspended in 10 ml 25 mM Tris-HCl, pH 7 anddialyzed overnight against the same buffer. The dialyzed sample wasloaded onto a DEAE-Sepharose column (Sigma) equilibrated with 25 mMTris-HCl, pH 7. After the column was washed with 200 ml of the samebuffer, the bound phytase was eluted with 1 M NaCl in 25 mM Tris-HCl, pH7. Three fractions exhibiting the highest phytase and acid phosphataseactivities were pooled and dialyzed against 25 mM Tris-HCl, pH 7.5overnight for the following studies.

Electrophoretic analysis. Protein concentration was measured by theLowry's method (21). Non-denaturing gel electrophoresis and SDS-PAGE(15%) were performed as described by Laemmli (22). Proteins in SDS-PAGEwere stained with Coomassie brillant blue R-250. Acid phosphatase orphytase activity in bands of the non-denaturing gel was detected asdescribed previously (17). After electrophoresis, the gel was incubatedfor 20 min at 25° C. in 0.2% α-19 naphtylphosphate (or sodium phytate),0.1% Fast Garnet GBC salts, 1 mM CaC12, and 0.5 M Tris-HCl buffer pH7.0.

Deglycosylation of the enzymes. Deglycosylation of r-appA2 was doneusing 0.3 IU of endoglycosidase H_(f) (Endo H_(f)) for 4 h at 7° C.according to the manufacturer instructions (New England Biolabs,Beverly, Mass.). The deglycosylated proteins were analyzed in a 15%SDS-PAGE as described above.

Enzyme properties and hydrolysis of phytate phosphorus in soybean meal.Phytase activity at different pH was determined at 33° C., using threedifferent buffers. The temperature optimum for each enzyme wasdetermined at its optimal pH. The K_(m) and V_(mas), values for r-appA2and r-appA were determined at the optimal pH of each enzyme and 37° C.Hydrolysis of phytate phosphorus by r-appA2 was compared with that ofr-appA and r-phyA. Different amounts of the purified enzymes wereincubated with 1 g soybean meal in a 5 mL buffer (10 mM HCl or 0.2 Mcitrate) at their respective optimal pH (2.5 for r-appA, 3.5 forr-appA2, and 5.5 for r-phyA) at 37° C. for 2 h. The released inorganicphosphorus was determined as previously described (25).Thermostabilities of these three enzymes were compared. Each of theenzymes (2 mg/ml) was diluted 1:200 in 0.2 M sodium citrate, pH 5.5, andincubated for 20 min at 25, 37, 55, 65, 80 and 100° C. The samples wereplaced on ice for 30 min and the remaining phytase activity was measuredat 37° C.

Statistical test employed The Mann-Withney U-test was used for all thestatistical evaluations (46).

Example 8 Bacterial Colony Screening and Identification

A total of 93 colonies were isolated. Over 70 colonies had intracellularphytase activity less than 500 U/g protein, and 6 colonies hadactivities greater than 1,000 U/g protein. Colony 88 demonstrated thehighest phytase activity (2,927 U/g protein), with an acid phosphataseactivity (1,391 U/g protein). Thus, it was chosen for furtherexperiments. The production of phytase and acid phosphatase activitiesby the colony was greater in Sweet E than LB broth and greater atanaerobic than aerobic conditions. Subsequently, the colony wasidentified as a gram negative E. coli. This was confirmed, inparticular, by the substrate fermentation profile.

Example 9 Cloning and Sequencing of the Pig E. coli appA2 Gene

A 1482 bp (whole) and a 1241 bp (coding region) fragments were amplifiedfrom the genomic DNA of Colony 88 (FIG. 6). Except for the E. coli appAgene and the Bacillus phytase gene, no significant sequence homologieswere found in the GenPro databank (version 61), GeneBank or EMBLdatabases using BLAST program. The whole nucleotide sequence had 47 and95% homology with the Bacillus sp. DS 11 phytase gene (GeneBankaccession number 3150039) and E. coli appA, respectively. In spite ofsuch a high nucleotide sequence homology, there were distinctdifferences between appA and appA2 and their encoding polypeptides.First, seven amino acids were different in the deduced peptidesequences: one in the signal peptide, L4F; six in the coding region,S102P, P195S, S197L, K202N, K298M, and T299A. Second, the 73 bpuntranslated region, located between the lead sequence and codingregion, was shorter by 6 bp than that of appA. However, the threeputative N-glycosylation sites were still located in the coding regionat the same place. The DNA fragment was sequenced for five times toverify these differences. Compared with phyA, appA2 had only a 19% ofamino acid sequence homology. The sequence has been transmitted toGeneBank data library with the accession number 250016.

Example 10 Expression of appA2 in Pichia Pastoris

A total of 42 transformants were analyzed for phytase and acidphosphatase activities at various intervals. Three days after methanolinduction, 13 transformants produced phytase activity from 18 to 114U/mL of medium and acid phosphatase activity from 7 to 42 U/mL.Meanwhile, 22 appA transformants expressed phytase activity from 25 to130 U/mL and acid phosphatase activity from 59 to 85 U/mL. The appA2transformant that demonstrated the highest activities was used in theexpression time course (FIG. 7) and other studies. The appA2 mRNA levelreached the peak at 4 h (FIGS. 7 and 8), remained high until 12 h, andthereafter declined significantly. No appA2 mRNA signal was detected inthe control cells. Both the extracellular phytase and acid phosphataseactivities produced by the transformant increased sharply between 0 and24 hours. Thereafter, the acid phosphatase activity remained nearlyunchanged while phytase activity increased much less over time than thatat the earlier phase.

Example 11 Characterization of the Purified Enzymes

The specific phytase activity of the purified r-appA2, r-appA, andr-phyA enzymes was 28.9, 30.7, and 19.8 U/mg protein, respectively. Thepurified r-appA2 demonstrated a higher affinity for sodium phytate thanpNNP, as shown by the K_(m) and V_(max) values (Table 1). When sodiumphytate was used as the substrate, the pH curve was significantlydifferent among the three enzymes.

TABLE 1 Kinetic parameters of the purified r-appA and r-appA2 expressedin Pichia pastoris r-appA r-appA2 K_(m), mM Sodium phytate 1.03 0.66p-NPP 2.26 1.43 V_(mzx), μmole min ⁻¹ mg⁻¹ Sodium phytate 89 117 p-NPP310 340

The pH optimum was between 2.5 and 3.5 for r-appA2, 2.5 for r-appA, and2.5 and 5.5 for r-phyA phytase (FIG. 9). However, the two E. colienzymes demonstrated the same pH optimum (2.5) for the substrate pNNP.In addition, both r-appA and r-appA2 had the same temperature optimum(55° C.) which was slightly lower than that of r-phyA. These two enzymesalso had very similar thermostabilities of phytase activity which wereslightly higher between 37 and 60° C. and lower between 65 and 100° C.than that of r-phyA. The acid phosphatase activity of r-appA2 thatremained after different temperature treatments was shown in thenon-denaturing gel, as a unique band of 71 kDa (FIG. 10). The activitywas largely or completely lost at 65 or 80° C., but somehow recoveredpartially at 100° C. When the three purified recombinant enzymes wereincubated with soybean meal, r-appA2 protein released significantly morephosphorus from phytate than the other two enzymes (FIG. 11).

Example 12 Effects of Deglycosylation on Enzyme Properties

After the three purified enzymes were treated with β-mercaptoethanol andEndo H_(f), more than 90% of their activities for both sodium phytateand pNNP were lost. But, Endo H_(f) alone had no significant effect ontheir catalytic activities. Deglycosylation of r-appA2 resulted in asingle band with an apparent Mr of 46.3 kDa from three distinguishedbands for the glycosylated forms with apparent Mr of 50.5, 53 and 56kDa. This gave a range of glycosylation for r-appA2 between 8.3 and17.3%.

In the above examples, a phytase-producing E. coli strain was isolatedfrom the pig colon content. Using primers based on the E. coli pH 2.5acid phosphatase gene (appA) described by Dassa et al. (18), a 1489 bpDNA fragment was amplified from the genomic DNA of the strain. Thisfragment, designated as appA2 (SEQ. ID. No. 9), encodes a protein of 433amino acids (SEQ. ID. No. 1) with 3 putative N-glycosylation sites. Thededuced peptide contains both the N-terminal motif (RHGXRXP, position:38-44) (SEQ. ID. No. 7) and the C-terminal motif (HD, position:325-326), characteristic for histidine acid phosphatases (8). Inaddition, there is a lead sequence of 30 amino acids (SEQ. ID. No. 8)and an untranslated region of 73 bp. Among the available sequencedatabases, only the E. coli appA pH 2.5 acid phosphatase and theBacillus sp. DS 11 phytase genes share some homology with appA2 (95% and47% in nucleotide sequence, respectively). In spite of the high homologybetween appA and appA2, there are distinct differences between these twogenes and their respective proteins. First, seven amino acids differbetween the two deduced polypeptide sequences: one within the signalpeptide and six in the coding region. Second, the 73 bp untranslatedregion between the lead sequence and the coding region was shorter by 6bp than that of appA. All those differences have been confirmed by fiverepetitive nucleotide sequencing analysis.

More importantly, when these two genes are transformed into the samehost, Pichia pastoris, the expressed proteins r-appA and r-appA2 showdifferently biochemical characteristics. Although both exhibit the samepH optimum of 2.5 for pNNP, r-appA2 has a broad pH optimum between 2.5and 3.5 while r-appA had it at 2.5 for sodium phytate. Compared withr-appA, the r-appA2 has a higher affinity for both substrates, as shownby the lower K_(m) and higher V_(max) values, and releases morephosphorus from phytate in soybean meal in vitro. Thus, the catalyticfunction of r-appA2, towards phosphorus hydrolysis from phytate orphosphate, seems to be better than that of r-appA. Apparently, the sixamino acid exchanges in the polypeptide may not be not just apolymorphism of the enzyme, but rather responsible for the observedkinetic differences. Thus, it seems reasonable to state that the appA2is a different gene from appA, although a more defined structuralanalysis is needed to elucidate the relationship between specific aminoacid exchanges and functional alterations of these two enzymes. It willbe necessary to produce the crystal of both enzymes first for futurestructural studies (27).

Previously, several E. coli enzymes have been reported to hydrolyze pNNPor sodium phytate (18, 19, 39-41). Greiner et al. (39) characterized twoE. coli phytases (P1 and P2). They found that the purified E. coliphytase P2 shares a great identity with the E. coli pH 2.5 acidphosphatase in the N-terminal sequence, chemical properties, andkinetics. Thus, they suggested that these two enzymes might be the sameprotein and the E. coli pH 2.5 acid phosphatase should better beregarded as a phytase. Indeed, both r-appA acid phosphatase and r-appA2are not only able to hydrolyze phytate in the pure chemical form or inthe natural food, but also have a higher affinity for sodium phytatethan pNNP. Therefore, these two enzymes could be classified as phytases.

Compared with the purified phytase P2 (39), r-appA2 has the same optimumtemperature (55° C.) and similar molecular mass after deglycosylation(46.3 kDa). Based on the SDS-PAGE and non-denaturing gel analyses, theprotein is also monomeric. However, r-appA2 has a more acidic pH optimum(2.5 to 3.5 vs. 4.5 for P2) and contains 8 to 14% of sugar moietiesbecause of the N-glycosylation in Pichia. Deglycosylation of r-appA2with Endo H_(f) reduces the molecular size but has a minimal effect onits activity. In contrast, when the protein is incubated withP-mercaptoethanol and Endo Hf, the phytase and acid phosphataseactivities of r-appA2 are considerably reduced. This indicates thatdisulfide bonds are required for its phytase activity as previouslyshown for the A. ficuum phytase (47).

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

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9 1 433 PRT Escherichia coli UNSURE (433) Xaa at position 433 in thissequence is unknown 1 Met Lys Ala Ile Leu Ile Pro Phe Leu Ser Leu LeuIle Pro Leu Thr 1 5 10 15 Pro Gln Ser Ala Phe Ala Gln Ser Glu Pro GluLeu Lys Leu Glu Ser 20 25 30 Val Val Ile Val Ser Arg His Gly Val Arg AlaPro Thr Lys Ala Thr 35 40 45 Gln Leu Met Gln Asp Val Thr Pro Asp Ala TrpPro Thr Trp Pro Val 50 55 60 Lys Leu Gly Trp Leu Thr Pro Arg Gly Gly GluLeu Ile Ala Tyr Leu 65 70 75 80 Gly His Tyr Gln Arg Gln Arg Leu Val AlaAsp Gly Leu Leu Ala Lys 85 90 95 Lys Gly Cys Pro Gln Pro Gly Gln Val AlaIle Ile Ala Asp Val Asp 100 105 110 Glu Arg Thr Arg Lys Thr Gly Glu AlaPhe Ala Ala Gly Leu Ala Pro 115 120 125 Asp Cys Ala Ile Thr Val His ThrGln Ala Asp Thr Ser Ser Pro Asp 130 135 140 Pro Leu Phe Asn Pro Leu LysThr Gly Val Cys Gln Leu Asp Asn Ala 145 150 155 160 Asn Val Thr Asp AlaIle Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp 165 170 175 Phe Thr Gly HisArg Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu 180 185 190 Asn Phe SerGln Leu Asn Leu Cys Leu Asn Arg Glu Lys Gln Asp Glu 195 200 205 Ser CysSer Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala 210 215 220 AspAsn Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr 225 230 235240 Glu Ile Phe Leu Leu Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp 245250 255 Gly Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His260 265 270 Asn Ala Gln Phe Tyr Leu Leu Gln Arg Thr Pro Glu Val Ala ArgSer 275 280 285 Arg Ala Thr Pro Leu Leu Asp Leu Ile Met Ala Ala Leu ThrPro His 290 295 300 Pro Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro ThrSer Val Leu 305 310 315 320 Phe Ile Ala Gly His Asp Thr Asn Leu Ala AsnLeu Gly Gly Ala Leu 325 330 335 Glu Leu Asn Trp Thr Leu Pro Gly Gln ProAsp Asn Thr Pro Pro Gly 340 345 350 Gly Glu Leu Val Phe Glu Arg Trp ArgArg Leu Ser Asp Asn Ser Gln 355 360 365 Trp Ile Gln Val Ser Leu Val PheGln Thr Leu Gln Gln Met Arg Asp 370 375 380 Lys Thr Pro Leu Ser Leu AsnThr Pro Pro Gly Glu Val Lys Leu Thr 385 390 395 400 Leu Ala Gly Cys GluGlu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala 405 410 415 Gly Phe Thr GlnIle Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu 420 425 430 Xaa 2 21 DNAEscherichia coli 2 ggaattccag agtgagccgg a 21 3 22 DNA Escherichia coli3 ggggtacctt acaaactgca cg 22 4 22 DNA Escherichia coli 4 taaggagcagaaacaatgtg gt 22 5 21 DNA Escherichia coli 5 ggaattccag agtgagccgg a 216 22 DNA Escherichia coli 6 ggggtacctt acaaactgca cg 22 7 7 PRTArtificial Sequence Description of Artificial Sequence N-terminal motif7 Arg His Gly Xaa Arg Xaa Pro 1 5 8 30 PRT Escherichia coli 8 Met TrpTyr Phe Leu Trp Phe Val Gly Ile Leu Leu Met Cys Ser Leu 1 5 10 15 SerThr Leu Val Leu Val Trp Leu Asp Pro Arg Leu Lys Ser 20 25 30 9 1489 DNAEscherichia coli 9 taaggagcag aaacaatgtg gtatttcctt tggttcgtcggcattttgtt gatgtgttcg 60 ctctccaccc ttgtgttggt atggctggac ccgcgattgaaaagttaacg aacgtaagcc 120 tgatccggcg cattagcgtc gatcaggcaa taatatcggatatcaaagcg gaaacatatc 180 gatgaaagcg atcttaatcc catttttatc tcttttgattccgttaaccc cgcaatctgc 240 attcgctcag agtgagccgg agctgaagct ggaaagtgtggtgattgtca gccgtcatgg 300 tgtgcgtgcc ccaaccaagg ccacgcaact gatgcaggatgtcaccccag acgcatggcc 360 aacctggccg gtaaaactgg gttggctgac accacgcggtggtgagctaa tcgcctatct 420 cggacattac caacgccagc gtctggtggc cgacggattgctggcgaaaa agggctgccc 480 gcagcctggt caggtcgcga ttattgctga tgtcgacgagcgtacccgta aaacaggcga 540 agccttcgcc gccgggctgg cacctgactg tgcaataaccgtacataccc aggcagatac 600 gtccagtccc gatccgttat ttaatcctct aaaaactggcgtttgccaac tggataacgc 660 gaacgtgact gacgcgatcc tcagcagggc aggagggtcaattgctgact ttaccgggca 720 tcggcaaacg gcgtttcgcg aactggaacg ggtgcttaatttttcccaat taaacttgtg 780 ccttaaccgt gagaaacagg acgaaagctg ttcattaacgcaggcattac catcggaact 840 caaggtgagc gccgacaatg tttcattaac cggtgcggtaagcctcgcat caatgctgac 900 ggaaatattt ctcctgcaac aagcacaggg aatgccggagccggggtggg gaaggatcac 960 tgattcacac cagtggaaca ccttgctaag tttgcataacgcgcaatttt atttactaca 1020 acgcacgcca gaggttgccc gcagtcgcgc caccccgttattggatttga tcatggcagc 1080 gttgacgccc catccaccgc aaaaacaggc gtatggtgtgacattaccca cttcagtgct 1140 gtttattgcc ggacacgata ctaatctggc aaatctcggcggcgcactgg agctcaactg 1200 gacgcttcca ggtcagccgg ataacacgcc gccaggtggtgaactggtgt ttgaacgctg 1260 gcgtcggcta agcgataaca gccagtggat tcaggtttcgctggtcttcc agactttaca 1320 gcagatgcgt gataaaacgc cgctatcatt aaatacgccgcccggagagg tgaaactgac 1380 cctggcagga tgtgaagagc gaaatgcgca gggcatgtgttcgttggccg gttttacgca 1440 aatcgtgaat gaagcgcgca taccggcgtg cagtttgtaatggtacccc 1489

What is claimed is:
 1. An isolated AppA2 polypeptide having improved phytase activity and having an amino acid sequence as shown in SEQ. ID No.
 1. 2. The isolated polypeptide according to claim 1, wherein the polypeptide is expressed in yeast.
 3. The isolated polypeptide according to claim 2, wherein the yeast is Pichia pastoris.
 4. The isolated polypeptide according to claim 2, wherein the yeast is Saccharomyces cerevisiae.
 5. The isolated polypeptide according to claim 1, wherein the polypeptide is encoded by a recombinant gene comprising: a promoter; a coding region encoding the polypeptide according to claim 1; and a terminator.
 6. An improved animal feed comprising the polypeptide according to claim
 1. 